A commercially available multiplex real-time PCR for detection of pathogens in cardiac valves from patients with infective endocarditis.

Infective endocarditis (IE) is a life-threatening condition, burdened by high mortality. Current guidelines recommend that, in case of negative culture result, tissues from excised heart valves or vegetations from patients with suspected IE should be referred for broad-range bacterial PCR and sequencing. In this proof-of-concept study, the diagnostic utility of the commercially available multiplex real-time PCR system SeptiFast (SF), performed on cardiac valves, was evaluated in a selected population of 20 patients with definite IE of known origin, in comparison with culture. A significant difference was found between SF and culture in the rate of pathogen detection (19 versus 3 respectively; chi-square 14.06; P=0.0002). SF sensitivity was 95%; specificity, 100%; positive predictive value (PPV), 100%; and negative predictive value (NPV), 83.3%. Culture sensitivity was 15%; specificity, 100%; PPV, 100%; and NPV, 22.7%. SF assay, performed on culture-negative excised heart valves, can be useful for the etiological diagnosis of IE.

A prediction model for real-time PCR results in blood samples from febrile patients with suspected sepsis.

Sepsis, a systemic, deleterious host response to infection that leads to organ dysfunction, is a potentially deadly condition needing prompt identification of the causative organisms and early appropriate antimicrobial therapy. Among non-culture-based diagnostic methods, SeptiFast (SF) can be employed to speed bacterial and fungal DNA detection, but it suffers from poor sensitivity and high cost. The aim of the present study, performed in 285 febrile patients, was to develop a prediction model to restrict the SF assay to clinical cases with a high probability of positive SF results. The prevalence of SF results positive for a pathogen was 17.2 %. Independent predictors of positive results were: blood sampling within 12 h after the onset of fever [odds ratio (OR) 20.03; 95 % confidence interval (CI) 6.87-58.38; P<0.0001]; ≥0.5 ng serum procalcitonin (PCT) ml(-1) (OR 18.52; 95 % CI 5.12-67.02; P<0.0001); body temperature ≥38 °C (OR 3.78; 95 % CI 1.39-10.25; P = 0.009); ≤3 g serum albumin dl(-1) (OR 3.40; 95 % CI 1.27-9.08; P = 0.014); and ≥13 000 white blood cells mm(-3) (OR 2.75; 95 % CI 1.09-7.69; P = 0.05). The model showed good calibration (Hosmer-Lemeshow chi-squared 1.61; P = 0.978). Area under the receiving operating characteristic curve was 0.944 (95 % CI 0.914-0.973; P<0.0001). These results suggest that a prediction model based on PCT and a few other routinely available laboratory and clinical variables could be of help in selecting patients with a high probability of SF-positive results.

Microbial and vaginal determinants influencing Mycoplasma hominis and Ureaplasma urealyticum genital colonization in a population of female patients.

Mycoplasma hominis and Ureaplasma urealyticum are associated with chorioamnionitis, preterm delivery and pelvic inflammatory disease. The aim of this study was to evaluate the possible risk factors of co-colonization by M. hominis in patients already colonized by U. urealyticum and compare demographic parameters, vaginal pH and microbiota of women colonized by U. urealyticum or M. hominis. A total of 452 patients positive for U. urealyticum or M. hominis were analysed, 421 (93.1%) of whom were positive for U. urealyticum and 31 (6.9%) for M. hominis. Patients positive for M. hominis compared to patients positive for U. urealyticum were more frequently colonized by Gardnerella vaginalis (71% vs 18.5%; p 0.0001), less frequently by lactobacilli (16.1% vs 61.5%; p 0.0001), and more frequently had a pH value higher than 4.5 (96.8% vs 57%; p 0.0001), all conditions associated to bacterial vaginosis (BV). Logistic regression analysis showed that only G. vaginalis colonization and pH higher than 4.5 were independently related to M. hominis colonization (respectively p 0.0001 and p 0.016). Thus, in women colonized by U. urealyticum, BV is an independent risk factor for M. hominis co-colonization.

Fatal Nocardia farcinica Bacteremia Diagnosed by Matrix-Assisted Laser Desorption-Ionization Time of Flight Mass Spectrometry in a Patient with Myelodysplastic Syndrome Treated with Corticosteroids.

Nocardia farcinica is a Gram-positive weakly acid-fast filamentous saprophytic bacterium, an uncommon cause of human infections, acquired usually through the respiratory tract, often life-threatening, and associated with different clinical presentations. Predisposing conditions for N. farcinica infections include hematologic malignancies, treatment with corticosteroids, and any other condition of immunosuppression. Clinical and microbiological diagnoses of N. farcinica infections are troublesome, and the isolation and identification of the etiologic agent are difficult and time-consuming processes. We describe a case of fatal disseminated infection in a patient with myelodysplastic syndrome, treated with corticosteroids, in which N. farcinica has been isolated from blood culture and identified by Matrix-Assisted Laser Desorption-Ionization Time of Flight Mass Spectrometry. The patient died after 18 days of hospitalization in spite of triple antimicrobial therapy. Nocardia farcinica infection should be suspected in patients with history of malignancy, under corticosteroid therapy, suffering from subacute pulmonary infection,and who do not respond to conventional antimicrobial therapy. Matrix-Assisted Laser Desorption-Ionization Time of Flight Mass Spectrometry can be a valuable tool for rapid diagnosis of nocardiosis.

Rapid identification of bacterial and fungal pathogens from positive blood cultures by MALDI-TOF MS.

Sepsis is a syndrome characterized by a systemic inflammatory response due to severe infection. Early detection of causal agents and appropriate antimicrobial treatment reduce mortality. Conventional microbiological methods often do not provide time critical results for an optimal early management. We used an in-house protocol based on Tween 80 to process 109 positive blood cultures for bacteria and yeast identification by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS), and results were compared to standard reference or automated methods. MALDI-TOF MS correctly identified 91.7% of the isolates. Correct identification was obtained for 57/62 (91.9%) aerobic/facultative anaerobic Gram-positive isolates, 53 (85.5%) at species level, and 4 (6.4%) at the genus level; 32/32 (100%) aerobic/facultative anaerobic Gram-negative isolates, 31 (96.9%) at species level, and 1 (3.1%) at the genus level; 7/7 (100%) obligate anaerobes, all at the genus level; 3/7 (42.8%) fungi, all at genus level. Overall, the median identification time of MALDI-TOF MS vs reference standard methods was significantly shorter: median (interquartile range) 7.1h (4.7-10.2) vs 48.1h (32.5-50.0), p<0.0001. MALDI-TOF MS is a valuable tool for rapid identification of pathogens in septic patients. An in-house protocol based on Tween 80 can be used to process positive blood cultures.

A novel bioluminescence mouse model for monitoring oropharyngeal candidiasis in mice.

Oropharyngeal Candida albicans (C. albicans) infection usually occurs in patients with altered cell-mediated immune response. Many animal models have been developed for studying the pathogenesis of disease. Here we describe a new model for real-time monitoring of oral candidiasis. Mice were rendered susceptible to oral candidiasis by injection with cortisone acetate. Oral infection was performed by placing a swab saturated with genetically engineered bioluminescent strain of C. albicans sublingually. An in vivo imaging technique, exploiting stably trasformed C. albicans that costitutively express luciferase, was adopted. This novel longitudinal study represents a powerful tool to: (1) test real-time progression of infection, (2) identify the target site of C. albicans in specific organs, (3) evaluate the efficacy of antifungal therapies and (4) explore the spread of C. albicans from the local to systemic compartment in a new way.

Secreted aspartic proteases of Candida albicans activate the NLRP3 inflammasome.

In a recent report, we demonstrated that distinct members of the secreted aspartic protease (Sap) family of Candida albicans are able to induce secretion of proinflammatory cytokines by human monocytes, independently of their proteolytic activity and specific pH optima. In particular, C. albicans Sap2 and Sap6 potently induced IL-1ß, TNF-α, and IL-6 production. Here, we demonstrate that Sap2 and Sap6 proteins trigger IL-1ß and IL-18 production through inflammasome activation. This occurs via NLRP3 and caspase-1 activation, which cleaves pro-IL-1ß into secreted bioactive IL-1ß, a cytokine that was induced by Saps in monocytes, in monocyte-derived macrophages and in dendritic cells. Downregulation of NLRP3 by RNA interference strongly reduced the secretion of bioactive IL-1ß. Inflammasome activation required Sap internalization via a clathrin-dependent mechanism, intracellular induction of K(+) efflux, and ROS production. Inflammasome activation of monocytes induced by Sap2 and Sap6 differed from that induced by LPS-ATP in several aspects. Our data reveal novel immunoregulatory mechanisms of C. albicans and suggest that Saps contribute to the pathogenesis of candidiasis by fostering rather than evading host immunity.

Mouse strain-dependent differences in estrogen sensitivity during vaginal candidiasis.

The animal models available for studying the immune response to genital tract infection require induction of a pseudo estrous state, usually achieved by administration of 17-ß-estradiol. In our experimental model of vaginal candidiasis, under pseudo estrus, different strains of mice were used. We observed major differences in the clearance of Candida albicans infection among the different strains, ascribable to differing susceptibility to estradiol treatment. In the early phase of infection CD1, BALB/c, C57BL/6 albino and C57BL/6 mice were colonized to similar levels, while in the late phase of infection, BALB/c mice, which are considered genetically resistant to C. albicans infection, exhibited greater susceptibility to vaginal candidiasis than CD1 and C57BL/6 albino strains of mice. This was because estradiol induced "per se" enlarged and fluid-filled uteri, more pronounced in infected mice and consistently more evident in BALB/c and C57BL/6 mice than in CD1 mice. Unlike CD1, BALB/c and C57BL/6 mice showed a heavy fungal colonization of the uterus, even though C57BL/6 mice apparently cleared C. albicans from the vagina. The presence of C. albicans in the vagina and uterus was accompanied by a heavy bacterial load. Collectively these observations prompted us to carry out a careful analysis of estradiol effects in a mouse model of vaginal infection.

Typing of nosocomial outbreaks of Acinetobacter baumannii by use of matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been evaluated for the identification of multidrug-resistant Acinetobacter baumannii nosocomial outbreaks in comparison with the repetitive sequence-based PCR DiversiLab system. The results suggest that MALDI-TOF MS can be used for real-time detection of Acinetobacter outbreaks before results from DNA-based systems are available.

Inflammation in aspergillosis: the good, the bad, and the therapeutic.

Aspergillosis includes a spectrum of diseases caused by different Aspergillus spp. New insights into the cellular and molecular mechanisms of resistance and immune tolerance to the fungus in infection and allergy have been obtained in experimental settings. The fact that virulence factors, traditionally viewed as fungal attributes, are contingent upon microbial adaptation to various environmental stresses encountered in the human host implies that the host and fungus are jointly responsible for pathogenicity. Ultimately, despite the occurrence of severe aspergillosis in immunocompromised patients, clinical evidence indicates that aspergillosis also occurs in the setting of a heightened inflammatory response, in which immunity occurs at the expense of host damage and pathogen eradication. Thus, targeting pathogenicity rather than microbial growth, tolerance rather than resistance mechanisms of defense may pave the way to targeted anti-inflammatory strategies in difficult-to-treat patients. The challenge now is to translate promising results from experimental models to the clinic.

Jack of all trades: thymosin α1 and its pleiotropy.

Thymosin α1 (Tα1), a thymosin-related 28-mer synthetic amino-terminal acetylated peptide, has gained increasing interest in recent years, due to its pleiotropy. The peptide has been used worldwide as an adjuvant or immunotherapeutic agent to treat disparate human diseases, including viral infections, immunodeficiencies, and malignancies. The peptide can enhance T cell, dendritic cell (DC), and antibody responses, modulate cytokine and chemokine production, and block steroid-induced apoptosis of thymocytes. Its central role in modulating DC function and activating multiple signaling pathways that contribute to different functions may offer a plausible explanation for its pleiotropic action. Additionally, the ability of Tα1 to activate the indoleamine 2,3-dioxygenase enzyme-which confers immune tolerance during transplantation and restrains the vicious circle of chronic inflammation-has been a turning point, suggesting a potential, specific function in immunity. Accordingly, Tα1 has recently been shown to promote immune reconstitution and improve survival of recipients of HLA-matched sibling T cell-depleted stem cell transplants in a phase I/II clinical trial. Thus, Tα1 continues to live up to its promises.

Antibody constant region peptides can display immunomodulatory activity through activation of the Dectin-1 signalling pathway.

We previously reported that a synthetic peptide with sequence identical to a CDR of a mouse monoclonal antibody specific for difucosyl human blood group A exerted an immunomodulatory activity on murine macrophages. It was therapeutic against systemic candidiasis without possessing direct candidacidal properties. Here we demonstrate that a selected peptide, N10K, putatively deriving from the enzymatic cleavage of the constant region (Fc) of human IgG(1), is able to induce IL-6 secretion and pIkB-α activation. More importantly, it causes an up-regulation of Dectin-1 expression. This leads to an increased activation of ß-glucan-induced pSyk, CARD9 and pIkB-α, and an increase in the production of pro-inflammatory cytokines such as IL-6, IL-12, IL-1ß and TNF-α. The increased activation of this pathway coincides with an augmented phagocytosis of non opsonized Candida albicans cells by monocytes. The findings suggest that some Fc-peptides, potentially deriving from the proteolysis of immunoglobulins, may cause an unexpected immunoregulation in a way reminiscent of innate immunity molecules.

DAMP signaling in fungal infections and diseases.

Fungal infections and diseases predominantly affect patients with deregulated immunity. Compelling experimental and clinical evidence indicate that severe fungal diseases belong to the spectrum of fungus-related inflammatory diseases. Some degree of inflammation is required for protection during the transitional response occurring temporally between the rapid innate and slower adaptive response. However, progressive inflammation worsens disease and ultimately prevents pathogen eradication. The challenge now is to elucidate cellular and molecular pathways distinguishing protective vs. pathogenic inflammation to fungi. In addition to fungal ligands of pattern recognition receptors (pathogen-associated molecular patterns, PAMPs), several host-encoded proteins, the damage-associated molecular patterns (DAMPs), are released during tissue injury and activate innate recognition receptors. DAMPs have been shown to regulate inflammation in fungal diseases. The DAMP/receptor for advanced glycation end-products axis integrated with the PAMP/Toll-like receptors axis in the generation of the inflammatory response in experimental and clinical fungal pneumonia. These emerging themes better accommodate fungal pathogenesis in the face of high-level inflammation seen in several clinical settings and point to DAMP targeting as a novel immunomodulatory strategy in fungal diseases.

Prevalence and antimicrobial susceptibility of Ureaplasma urealyticum and Mycoplasma hominis in a population of Italian and immigrant outpatients.

Ureaplasma urealyticum and Mycoplasma hominis are associated with non-gonococcal urethritis, increased risk of recurrent miscarriage, infertility and pelvic inflammatory disease. Migration flows from other countries change the local epidemiological profile of infectious diseases of patients treated by general practitioners and hospital doctors. Few studies have evaluated this ever-changing issue in the Italian population. The aim of this study was to assess possible differences in prevalence and antimicrobial susceptibility of U. urealyticum and M. hominis in a population of 433 Italian and immigrant outpatients by means of the commercially available MYCOFAST(®) Screening EvolutioN 3 Kit. Prevalence of positive samples was 44.5% in Italian patients and 53.4% in immigrants. Samples positive for U. urealyticum and total isolates were more frequent in African patients: U. urealyticum, 51.5% vs 33.3%; Yates-corrected chi-square=3.98; p=0.046; total isolates, 54.5% vs 34.3%; Yates-corrected chi-square=4.45; p=0.035. Among samples positive for U. urealyticum, 66.4% were resistant to ciprofloxacin, whereas 27.6% to ofloxacin. In M. hominis isolates, 66.7% were resistant to both azythromycin and roxythromycin. Our study showed how prevalence of genital mycoplasmas and antibiotic resistance profiles change in relation to the country of origin. Therefore, surveillance is critical for the early cure and prevention of the occurrence of resistant strains.

Immunity and tolerance to fungi in hematopoietic transplantation: principles and perspectives.

Resistance and tolerance are two complementary host defense mechanisms that increase fitness in response to low-virulence fungi. Resistance is meant to reduce pathogen burden during infection through innate and adaptive immune mechanisms, whereas tolerance mitigates the substantial cost of resistance to host fitness through a multitude of anti-inflammatory mechanisms, including immunological tolerance. In experimental fungal infections, both defense mechanisms are activated through the delicate equilibrium between Th1/Th17 cells, which provide antifungal resistance, and regulatory T cells limiting the consequences of the ensuing inflammatory pathology. Indoleamine 2,3-dioxygenase (IDO), a rate-limiting enzyme in the tryptophan catabolism, plays a key role in induction of tolerance against fungi. Both hematopoietic and non-hematopoietic compartments contribute to the resistance/tolerance balance against Aspergillus fumigatus via the involvement of selected innate receptors converging on IDO. Several genetic polymorphisms in pattern recognition receptors influence resistance and tolerance to fungal infections in human hematopoietic transplantation. Thus, tolerance mechanisms may be exploited for novel diagnostics and therapeutics against fungal infections and diseases.

Host defense pathways against fungi: the basis for vaccines and immunotherapy.

Fungal vaccines have long been a goal in the fields of immunology and microbiology to counter the high mortality and morbidity rates owing to fungal diseases, particularly in immunocompromised patients. However, the design of effective vaccination formulations for durable protection to the different fungi has lagged behind due to the important differences among fungi and their biology and our limited understanding of the complex host-pathogen interactions and immune responses. Overcoming these challenges is expected to contribute to improved vaccination strategies aimed at personalized efficacy across distinct target patient populations. This likely requires the integration of multifaceted approaches encompassing advanced immunology, systems biology, immunogenetics, and bioinformatics in the fields of fungal and host biology and their reciprocal interactions.

Roles of the amino terminal region and repeat region of the Plasmodium berghei circumsporozoite protein in parasite infectivity.

The circumsporozoite protein (CSP) plays a key role in malaria sporozoite infection of both mosquito salivary glands and the vertebrate host. The conserved Regions I and II have been well studied but little is known about the immunogenic central repeat region and the N-terminal region of the protein. Rodent malaria Plasmodium berghei parasites, in which the endogenous CS gene has been replaced with the avian Plasmodium gallinaceum CS (PgCS) sequence, develop normally in the A. stephensi mosquito midgut but the sporozoites are not infectious. We therefore generated P. berghei transgenic parasites carrying the PgCS gene, in which the repeat region was replaced with the homologous region of P. berghei CS (PbCS). A further line, in which both the N-terminal region and repeat region were replaced with the homologous regions of PbCS, was also generated. Introduction of the PbCS repeat region alone, into the PgCS gene, did not rescue sporozoite species-specific infectivity. However, the introduction of both the PbCS repeat region and the N-terminal region into the PgCS gene completely rescued infectivity, in both the mosquito vector and the mammalian host. Immunofluorescence experiments and western blot analysis revealed correct localization and proteolytic processing of CSP in the chimeric parasites. The results demonstrate, in vivo, that the repeat region of P. berghei CSP, alone, is unable to mediate sporozoite infectivity in either the mosquito or the mammalian host, but suggest an important role for the N-terminal region in sporozoite host cell invasion.

Diagnostic performance of a multiple real-time PCR assay in patients with suspected sepsis hospitalized in an internal medicine ward.

Early identification of causative pathogen in sepsis patients is pivotal to improve clinical outcome. SeptiFast (SF), a commercially available system for molecular diagnosis of sepsis based on PCR, has been mostly used in patients hospitalized in hematology and intensive care units. We evaluated the diagnostic accuracy and clinical usefulness of SF, compared to blood culture (BC), in 391 patients with suspected sepsis, hospitalized in a department of internal medicine. A causative pathogen was identified in 85 patients (22%). Sixty pathogens were detected by SF and 57 by BC. No significant differences were found between the two methods in the rates of pathogen detection (P = 0.74), even after excluding 9 pathogens which were isolated by BC and were not included in the SF master list (P = 0.096). The combination of SF and BC significantly improved the diagnostic yield in comparison to BC alone (P < 0.001). Compared to BC, SF showed a significantly lower contamination rate (0 versus 19 cases; P < 0.001) with a higher specificity for pathogen identification (1.00, 95% confidence interval [CI] of 0.99 to 1.00, versus 0.94, 95% CI of 0.90 to 0.96; P = 0.005) and a higher positive predictive value (1.00, 95% CI of 1.00 to 0.92%, versus 0.75, 95% CI of 0.63 to 0.83; P = 0.005). In the subgroup of patients (n = 191) who had been receiving antibiotic treatment for ≥24 h, SF identified more pathogens (16 versus 6; P = 0.049) compared to BC. These results suggest that, in patients with suspected sepsis, hospitalized in an internal medicine ward, SF could be a highly valuable adjunct to conventional BC, particularly in patients under antibiotic treatment.

Procalcitonin predicts real-time PCR results in blood samples from patients with suspected sepsis.

BACKGROUND: Early diagnosis and rapid bacterial identification are of primary importance for outcome of septic patients. SeptiFast® (SF) real-time PCR assay is of potential utility in the etiological diagnosis of sepsis, but it cannot replace blood culture (BC) for routine use in clinical laboratory. Procalcitonin (PCT) is a marker of sepsis and can predict bacteremia in septic patients. The aim of the present study was to investigate whether PCT serum levels could predict SF results, and could help screening febrile patients in which a SF assay can improve the etiological diagnosis of sepsis. METHODS: From 1009 febrile patients with suspected sepsis, 1009 samples for BC, SF real-time PCR, and PCT determination were obtained simultaneously, and results were compared and statistically analysed. Receiver operating characteristic (ROC) curves were generated to determine the area under the curve and to identify which cut-off of PCT value produced the best sensitivity to detect SF results. RESULTS: Mean PCT values of sera drawn simultaneously with samples SF positive (35.42 ± 61.03 ng/ml) or BC positive (23.14 ± 51.56 ng/ml) for a pathogen were statistically higher than those drawn simultaneously with SF negative (0.84 ± 1.67 ng/ml) or BC negative (2.79 ± 16.64 ng/ml) samples (p<0.0001). For SF, ROC analysis showed an area under the curve of 0.927 (95% confidence interval: 0.899-0.955, p<0.0001). The PCT cut-off value of 0.37 ng/ml showed a negative predictive value of 99%, reducing the number of SF assays of 53.9%, still identifying the 96.4% of the pathogens. CONCLUSION: PCT can be used in febrile patients with suspected sepsis to predict SF positive or negative results. A cut-off value of 0.37 ng/ml can be considered for optimal sensitivity, so that, in the routine laboratory activity, SF assay should not be used for diagnosis of sepsis in an unselected patient population with a PCT value <0.37 ng/ml.